Process for the preparation of gallic acid by co-culture

ABSTRACT

A process for the preparation of gallic acid using co-culture comprising providing a tannin-rich mixed substrate and a culture medium in fluid communication with each other, adding an induced innoculum comprising the fungi  Rhizopus oryzae  and  Aspergillus foetidus  to the substrate to obtain a fermented mass and gallic acid, extracting the gallic acid from the culture medium and the fermented mass using an organic solvent followed by evaporation of the organic solvent to obtain gallic acid.

FIELD OF INVENTION

The invention relates to a process for the preparation of gallic acid byco-culture.

BACKGROUND OF THE INVENTION

Enzymes are specific proteins of living tissues that act asbiocatalysts. Enzymes can accomplish those reactions at normaltemperatures and pressure which would otherwise require expensive,energy demanding high temperature and/or pressures, or might not bepossible at all. That is why, use of enzymes in industries is growing.

Many micro-organisms produce extra-cellular enzymes. They are chieflyhydrolasesand are involved primarily in the degradation ofmacromolecules to units capable of being taken into the living cell.With the versatility of micro-organisms in producing enzymes, newmethods of making many industrially important chemicals are beingexplored.

Current developments in biotechnology are yielding new applications formicrobial enzymes. In addition to the conventional applications in foodand fermentation industries, microbial enzymes have attained significantrole in biotransformations involving organic solvent media speciallybioactive compounds. Alongwith the use of micro-organisms to producebiomass and microbial metabolites, microbial cells may be used tocatalyse the conversion of a compound into a financially more valuablecompound. Microbial processes have the advantage of specificity over theuse of chemical reagents and of operating at relatively low pressuresand temperatures.

An important enzyme having various industrial applications is tannase(tannin acyhydrolase), which is an extra-cellular enzyme falling underthe hydrolase group of enzymes. Tannins are water-soluble, phenoliccompounds with molecular weight (500–3000) that have the property ofcombining with proteins, cellulose, gelatin and pectin and can beclassified into two distinct groups based on their structuralconfiguration, the hydrolysable tannins and the condensed tannins.

Tannase is widely used in the food and chemical industries. Further,tannase is used in wine making where it hydrolyses chlorogenic acid andquinic acid, which favourably influences taste in, the process of winemaking. Tannase also has potential in the manufacture of acorn wine.Tannase is also used alongwith lactase to treat grap juice and grapemusts

So as to remove phenolic substances for stabilization of the beverage.Tannase significantly reduces chill haze formation in beer.Disclouration and haze development during beer storage could beprevented by the enzymatic hydrolysis of wort phenolics with tannase.

Further, tannase is used in a preconversion treatment of fresh green teaflush in the production of instant tea. Tannase is also used in thedetermination of the structure of naturally occurring gallic acidesters.

Tannin acyl hydrolase commonly called tannase catalyzes, the hydrolysisof ester and depside bonds in such hydrolysable tannins as tannic acid,thereby releasing glucose and gallic acid. Gallic acid(3,4,5-Trihydroxybenzoic acid) finds various uses. In the pharmaceuticalindustry, gallic acid is used in the manufacture of trimethoprim. It isan antibacterial and is administered jointly with sulphonamide andtogether provide a broad spectrum action for medical treatment. TMPinhibits dihydrofolate reductase thereby blocking transformation ofdihydrofolate to tetrahydrofolate. The consumption co-efficient ofgallic acid in the manufacture of trimethoprim is 4.8. In the tanneryindustry gallic acid is used for homogenization of tannins, for thepreparation of high grade leather tannins. Gallic acid is also used inthe manufacture of ordinary writing inks and dyes, as a photographicdeveloper. It also finds use in the enzymatic synthesis of gallic acidesters like propyl gallate, which is mainly used as an antioxidant infats and oils, and also in beverages. It is used in the testing ofmineral acids, dihydroxy acetone, and alkaloids, and as a syntheticintermediate for the production of pyrogallol which is used for theproduction of pyrogallol which is used for staining fur, leather, hair,etc.

Use of various fugal species to produce tannase is known in the art. Anumber of micro-organisms including bacteria like (Bacillus pumilis, B.polymyxa, Corynebacterium sp., Klebsiella pneumoniae) fungi (Ascochytasp. Penicillium sp.) & yeasts (Candida sp.) have been reported toproduce tannase.

Production of tannase by a strain of Aspergillus niger was reported byTourrat et al. They found tannase activity was maximum when fermentationwas carried out in submerged culture at a constant air flow. The enzymeactivity was determined by gas chart method and the maximum enzymeactivity was reported to be 5.5 n Kat/ml. R. Banerjee et.al, hasreported the activity of tannase biosynthesis by a newly isolated R.oryzae. The experimental conditions were optimized in shake flaskcultures. Maximum enzyme activity was found to be 6.12 U/ml.

R. Banerjee et al have also reported the production of tannase by solidstate fermentation using R. oryzae.

Production of extracellular tannase by bacterial strains (B. polymyxa,B. puminis, klebsiclla pnon & Corynebacterium) within few hours ofculture with simultaneous release of gallic acid & glucose has beenreported by Deschamp et al.

Raj Kumar et al. have reported the isolation, purification and someproperties of Penicillium chrysogcnum tannase.

The enzyme is stable up to 30 C. and within the pH range of 4.0–6.0 Kmvalue was found to be 0.48×10⁻⁴M with tannic acid as the substrate.Metal salts at 20 Mm inhibited with enzyme.

Continuous production of gallic acid from tara tannin in a bioreactorusing Penicilium chrysogenum immobilized on sodium alginate & CaCl₂ isknown from Yamada et al. They have also reported the use of tannase inwine making industry.

Not much is known about the biotransformation of tannins to gallic acid.The literature available mainly relates to chemical process wherein theyield of gallic acid is was very low. Kar, B. et al have reported thebiotransformation of tannins to gallic acid by SSF and SmF process usinggallo seed cover and Rhizopus oryzae as the raw material.

However, the processes of the art suffer from various drawbacks ie theyrequire a long time and the yield reported is also low.

OBJECTS OF THE INVENTION

It is therefore an object of this invention to propose a process for thepreparation of gallic acid using mixed culture process, which requiresless time compared to conventional techniques.

It is a further object of this invention to propose a process for thepreparation of gallic acid using mixed culture technique which givesbetter yield and employs a novel substrate.

DESCRIPTION OF THE INVENTION

Thus according to this invention is provided a process for thepreparation of gallic using co-culture, comprising providing atannin-rich mixed substrate and a culture medium in fluid communicationwith each other, adding an induced innoculum comprising the fungiRhizopus oryzae and Aspergillus foetidus to the substrate to obtain afermented mass and gallic acid, extracting the gallic acid from theculture medium and the fermented mass using an organic solvent followedby evaporation of the organic solvent to obtain gallic acid.

In accordance with this invention, the raw materials used as mixedsubstrates are myrobalan fruit powder (Terminalia chebula, seedlesstype, PUNJAB variety) and gallo seed cover powder (Caesalpinia digyna)in a proportion of 1:3 to 3:1. These are substrates rich in tannin.Myrobalan seed contains 33–45% tannin and gallo seed cover containsabout 58% tannin. Two strains of the filamentous fungi Rhizopus oryzae(RO IIT RB13,NRRL-21498) and Aspergillus foetidus (GMRB013), isolatedfrom the soil of IIT, Kharagpur Campus have been used. The organisms aremaintained on 2% malt extract agar slant. A subculture of themicro-organisms is done on slants. A malt extract agar (MEA) medium issterilized and a portion of it is transferred into sterile test tubesand allowed to cool in slanting position.

After solidification, the sterile slants are inoculated with purecultures of Rhizopus oryzae (RO III RB13), NRRL-21498) and Aspergillusfoetidus (GMRR 013, MTCC 3557) and kept in an incubator. The slantcultures are then used for further work or stored. As tannase is anadaptive enzyme, a pre-induced innoculum for the culture is preparedwherein tannic acid in modified Czapekdox medium is sterilized andinoculated with a spore suspension prepared from the cultured slants.They are then kept in a BOD incubator under shaking, to produce theinduced innoculum. For subsequent studies of MSSF (Modified Solid StateFermentation), this induced innoculum is used.

The two microorganisms were used together where the ratio of theorganisms varied from 0.5:2 to 1:1, where the optimum ratio was 1:1. Itwas found in general, that upon carrying out the co-culture technique,the tannase and gallic acid produced was higher than that produced underpure culture conditions, and also the incubation period required wasshorter.

Fermentation is carried out in batch process under modified solid statefermentation in a modified tray type reactor. The raw materials used asmixed substrates are myrobalan fruit powder and gallo seed cover powdertaken in a proportion of 1:3 to 3:1. This substrate mixture is placed onthe float of the tray reactor. The tray reactors ordinarily used aremodified by providing a perforated float. This offers the uniqueadvantages of greater heat dissipation during fermentation whichprevents the biomass from getting denatured. Liquid modified Czapekdoxmedium in the ratio of 0.2:1 to 0.8:1 (solid-liquid ratio) is takenbeneath the float in the tray. Czapekdox medium is modified by usingtannic acid as the carbohydrate source, instead of glucose. Thus, themixed substrate placed on the float comes in contact with the liquidmedium in the tray. The modified tray reactor is then autoclaved.Fermentation of the substrates on the float is carried out by adding anappropriate amount of induced innoculum of Rhizopus oryzae andAspergillus foetidus, ie by co-culture method about 1×10⁵ to 2×10⁸spores per ml, preferably 2×10⁶ spores per ml is added to 20 gms of thesubstrate. The micro-organizm added to the substrate converts thesubstrate into the desired product, which leaches into the liquidmedium. The fermented material is removed, water is added and heated. Itis then cooled to room temperature and gallic acid is extracted using anorganic solvent such as diethyl ether and ethyl acetate.

The optimum conditions of pH, temperature, humidity and the effects ofquantity of innoculum, particle size and moisture on the production ofgallic acid have been evaluated.

The invention will now be explained in greater detail with the help ofthe following non-limiting examples:

EXAMPLE

Isolation of Tannase Producing Microorganisms

Two strains of filamentous fungi Rhizopus oryzae (RO IIT RB 13,NRRL-21498) and Aspergillus foetidus (GMRB013) were isolated from thesoil of I.I.T., Kharagpur campus by a baiting technique. The organismswere maintained on 2% malt extract agar slant.

Subculture of Microorganisms on Slants

100 ml of malt extract agar (MEA) medium was sterilized. Aftersterilization, 5 ml of it was transferred into sterile test tubes andallowed to cool in slanting position. After solidification, the sterileslants were inoculated with pure cultures of Rhizopus oryzae RO lITRB13, NRRL-21498) and Aspergillus foetidus (GMRB 013) in a 1:1 ratio bystreaking and was kept in incubator at 30° C. for (72–96) hrs.

The slant cultures were then used for further work or stored in fridgeat 4° C.

Preparation of Induced Innoculum

Tannase being an adaptive enzyme, pre-induced inoculum is required to beprepared. 50 ml of 2% tannic acid in Czapekdox medium was taken in 100ml conical flasks. It was then sterilised at 121° C. for 15 mins andinoculated with 2 ml of spore suspension prepared from the culturedslants. They were then kept in BOD incubator at 30° C. under shakingcondition for 72 hrs. For subsequent studies of SSF, this inducedinoculum was used.

Modified Solid State Fermentation Using Mixed Substrates by Co-CultureMethod

Fermentation was carried out in batch process under modified solid statefermentation in a modified tray type reactor.

The raw materials used as mixed substrates where myrobalan seed powder(Terminalia chebula) and gallo seed cover powder (Caesalpinia digyna)taken in a proportion of 1:3 to 3:1. This substrate mixture was placedon the float of the tray reactor. Liquid Czapek dox medium in the rationof 0.2:1 to 0.8:1 (solid-liquid ratio) is taken beneath the float in thetray. This kind of float with the substrate is kept above the liquid inthe tray in order to carry out modified solid state fermentation. Thus,the mixed substrate placed on the float comes in contact with the liquidmedium in the tray. The modified tray reactor is then autoclaved at 121°C. for 15 mins. Fermentation of the substrates on the float is carriedout by adding 2×10⁶ spores/ml of induced inoculum of Rhizopus oryzae andAspergillus foetidus per 20 gms of substrate ie. By co-culture method.The microorganism added to the substrate converts the substrate into thedesired product, which leaches into the liquid medium.

Gallic Acid Extraction

Gallic acid was isolated using the organic solvent ethyl acetate. Thefermented material was removed, water added, and heated to about(60–70)° C. because gallic acid is soluble at this temperature, and notin cold water. Then it was cooled to room temperature. Organic solventwas then added to it and the whole mixture was taken in a separatingfunnel. The mixture was immediately mixed thoroughly by vigorousshaking. Gallic acid being soluble in organic solvent, comes into theorganic phase and the rest of the matter remains in aqueous phase. Theaqueous phase was discarded and the organic layer was collected. Thisprocess was continued till the entire gallic acid came out into theorganic layer. The collected volume of organic layer was now taken forseparation of gallic acid from ethyl acetate in the rotary vacuumevaporator. The pressure and temperature at which this separation wasdone was 200 mbar and 70° C. Alternatively, the ethyl acetate layer wasextracted with diethyl ether, the ether layer was evaporated in a rotaryevaporator to obtain pure gallic acid. The yield of the gallic acidobtained was found to vary from 65.4 to 94.8%. Studies were conductedfor optimization of various environmental parameters for obtainingmaximum gallic acid production.

Optimization of Physicochemical Parameters for Tannase and Gallic AcidProduction by Modified Solid-State Fermentation

The effect of the various environmental parameters on the production oftannase and gallic acid upon carrying out MSSF by both the organismsusing mixed substrates under both pure culture and co-culture conditionswas studied

-   Effect of pH:—The effect of pH on production of tannase was studied    by varying the intitial pH of the Czapekdox medium from 3.5 to 7.0.    The optimum pH was found to be 5, at which tannase activity was 35.1    U/ml and gallic acid produced was 91.81%.-   Effect of temperature:—The effect of temperature on tannase and    gallic acid production was studied by varying the temperature in the    humidity cabinet from 25° C. to 40° C. where the optimum temperature    was found to be 30° C. The amount of tannase and gallic acid    produced was 36.4 U/ml and 93.25% respectively.-   Effect of humidity:—The effect of humidity on tannase production and    % yield of gallic acid was studied by varying the humidity in the    humidity chamber from 70% to 90% where the optimum lies between 80%.    The amount of tannase and gallic acid produced was 36.4 U/ml and    93.25% respectively.-   Effect of inoculum amount:—The optimum amount of inoculum required    for the maximum tannase production was obtained after varying the    amount of inoculum from 1 ml to 4 ml with optimum at 3 ml.-   Effect of moisture:—To study the effect of moisture on production of    tannase, addition of Czapekdox medium to myrobalan substrate was    varied from 0.2:1 to 0.8:1 where optimum lies to 0.4:1. Tannase and    gallic acid produced were 35.3 U/ml and 92.41 respectively at this    moisture condition. An incubation period of 48h was found to be the    optimum for Gallic acid production by co-culture method using mixed    substrates, giving a tannase activity of 33.1 U/ml.

A comparison between pure culture and co-culture methods shows the(following advantages of the co-culture method as highlighted in Table1.

Co-Culture with Pure Culture Rhizopus Pure Culture with AspergillusOryzae & with Rhizopus Foetidus Aspergillus Oryzae using using singleFoetidus Single Substrate Substrate Using mixed CONDITIONS (Myrobalam)(Myrobalam) Substrates 1) Temperature (25–40)° C. (25–40)° C. (25–40)°C. 2) PH 3.5–7 3.5–7 3.5–7 3) Humidity (70–90)% (70–90)% (70–90)% 4)Incubation 24–96 hrs 24–96 hrs 24–96 hrs period 5) Moisture 1:0.25–1:1.51:0.25–1:1.5 0.2:1–8:1 (Solid-Liquid Ratio) 6) Reactor Tray TrayModified Type Tray 7) Gallic Acid 44–87.67% 51.19–92.45% 65.42–94%

1. A process for the preparation of gallic acid comprising: providing atannin-rich mixed substrate and a culture medium, in a liquid medium;adding an induced innoculum comprising the fungi Rhizopus oryzae andAspergillus foetidus to said substrate to obtain a fermented mass andgallic acid; extracting gallic acid from the culture medium and thefermented mass using an organic solvent; and evaporating the organicsolvent to obtain gallic acid.
 2. The process as claimed in claim 1,wherein said tannin-rich mixed substrate comprises myrobalan fruitpowder (Terminalia chebula) and gallo seed cover powder (Caesalpiniadigyna) in a proportion of 1:3 to 1:⅓.
 3. The process as claimed inclaim 1, wherein said culture medium is a modified Czapek-Dox medium. 4.The process as claimed in claim 3, wherein the modified Czapek-Doxmedium comprises tannic acid as a carbohydrate source.
 5. The process asclaimed in claim 1, wherein the substrate and culture medium are presentin a ratio of 0.2:1 to 0.8:1.
 6. The process as claimed in claim 1,wherein the fungi are maintained on a 2% malt extract agar slant.
 7. Theprocess as claimed in claim 1, wherein the induced innoculum is obtainedby providing a tannic acid substrate in Czapek-Dox medium, sterilizingthe tannic acid substrate; and inoculating the substrate with a sporesuspension of the fungi Rhizopus oryzae and Aspergillus foetidus toobtain an inoculated substrate; and incubating the inoculated substrateto obtain an induced innoculum.
 8. The process as claimed in claim 1,wherein an induced innoculum containing 1×10 spores/ml to 2×10⁸ sporesper ml is added to 20 gins of the mixed substrate.
 9. The process asclaimed in claim 1, wherein the organic solvent is ethyl acetate. 10.The process as claimed in claim 1, which further includes the steps ofadding water to the culture medium and the fermented mass; heating at atemperature in the range of 60–70° C. to obtain an aqueous solution ofgallic acid, extracting the aqueous solution of gallic acid with anorganic solvent to obtain a solution of the gallic acid in the organicsolvent, followed by evaporation of the solvent to obtain gallic acid.11. The process as claimed in claim 10, wherein the organic solvent isethyl acetate.
 12. The process as claimed in claim 10, which furtherincludes an additional extraction step comprising using a seconddifferent solvent, to form a solution of gallic acid in said secondsolvent.
 13. The process as claimed in claim 12 wherein the seconddifferent solvent is ethyl ether.
 14. The process as claimed in claim 1,wherein the ratio of the fungi Rhizopus oryzae and Aspergillus foetidusranges from between 0.25:1 to 1:1.
 15. A process for the preparation ofgallic acid comprising: providing a tannin-rich mixed substrate and aculture medium, in a liquid medium, wherein said tannin-rich mixedsubstrate comprises myrabalan fruit powder (Terminalia chebula) andgallo seed cover powder (Caesalpinia digyna) in a proportion of 1:3 to1:⅓; adding an induced innoculum comprising the fungi Rhizopus oryzaeand Aspergilus foetidus to said substrate to obtain a fermented mass andgallic acid, wherein the ratio of the fungi Rhizopus oryzae andAspergillus foetidus ranges from 0.25:1 to 1:1; extracting gallic acidfrom the culture medium and the fermented mass using an organic solvent;and evaporating the organic solvent to obtain gallic acid.